Project Summary The bacteria in the Chlamydiales order are intracellular parasites of eukaryotic cells. They depend on a unique biphasic developmental cycle consisting of a replicative cell form and an infectious cell form. This developmental program alternates between two differentiated cell types, the elementary body (EB) and reticulate body (RB) and is critical to the completion of its life cycle. Within this order, the genus Chlamydia contains the causative agents of a number of important pathogens of humans. C. psittaci causes zoonotic infections resulting in pneumonia, while C. pneumoniae is a human pathogen that causes respiratory disease and is linked to atherosclerosis. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as the sexually transmitted disease Chlamydia. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and biphasic developmental cycle. The cell type specific division of labor (replication=RB, cell invasion=EB) in these pathogens generates a developmental cycle that results in a viral like one step growth curve with a defined eclipse period when no infectious progeny are present. Chlamydial pathogenesis is dependent on balancing the need to replicate with the need to create infectious progeny. It is not clear how this developmental process is regulated. In this proposal we aim to first Determine the molecular mechanisms that control RB to EB development. To uncover genes involved in regulating the cycle we will employ a forward genetic screen leveraging chemical mutagenesis and live cell imaging using novel reporter constructs to visualize the developmental cycle in real time. Our previous work uncovered a regulatory circuit involved in EB formation. The small non coding RNA, IhtA, inhibits the translation of the EB nucleoid factor and histone homolog HctA. By determining the mechanism of this regulation a second protein regulated by IhtA was discovered, the hypothetical protein CTL0322. Preliminary evidence indicates that CTL0322 is a DNA binding protein and is involved in RB to EB differentiation. The second aim will focus on Determining the role of the DNA binding protein CTL0322 in the chlamydial developmental cycle. To understand the role of CTL0322, we will employ a combination of genetics, isolation of suppressor mutants, biochemistry and pull down assays to identify binding partners, and transcriptional profiling to determine pathways impacted byCTL0322. Ultimately, understanding the process involved in regulating the unique chlamydial developmental cycle will lead to new narrowly targeted therapeutic targets helping to alleviate the burden of broad antimicrobial resistance.